Malignant cells are characterized by the extracellular production of superoxide anions by membranous NADPH oxidase (NOX-1). Here, the activity of NOX is controlled by oncogenes such as e.g., RAS by involving RAC. The superoxide anions generated by malignant cells and their dismutation product hydrogen peroxide are essential for the proliferation of these cells and for the maintenance of their transformed state (summarized in Heinzelmann and Bauer, [2010], Biol. Chem., Vol. 391, pp. 675-693). However, the other side of the coin from the extracellular production of reactive oxygen species (ROS) is the formation of intercellular ROS-mediated signal paths selectively directing against cells with the transformed phenotype. These are the main paths shown in FIG. 1, namely the HOCl and the NO/peroxynitrite signal path as well as two further paths of secondary importance, namely the nitryl chloride path and the metal catalyzed Haber-Weiss reaction that are not considered in FIG. 1. In the course of the tumor progression tumor cells acquire resistance against the intercellular ROS signal paths by expressing catalase (CAT) on their cell membrane. This inhibits the HOCl and the nitryl chloride path as well as the metal catalyzed Haber-Weiss reaction by converting hydrogen peroxide into water and oxygen, and counteracts the NO/peroxynitrite path by decomposing peroxynitrite and oxidizing NO to NO2 with the help of its active intermediate CAT FeIV=O+ (compound I), and so prevents the formation of peroxynitrite. Cancelling the catalase (CAT) mediated protection of tumor cells represents an attractive concept for the development of a novel form of tumor therapy that is specifically directed against cells with the malignant phenotype (due to the features of membranous catalase and superoxide anion production) and does not endanger normal cells, since these neither produce extracellular superoxide anions nor express membranous catalase.
The procedure disclosed in DE 103 58 077 A1 2005.07.28 permits search for active ingredients that specifically and directly inhibit membranous catalase and thus, reactivate intercellular ROS signal paths which then induce apoptosis in tumor cells. FIG. 2 schematically shows the ROS (reactive oxygen species) controlled induction of apoptosis that is reactivated by the inhibition of the catalase.
The same selective induction of apoptosis in tumor cells can be achieved when extracellular singlet oxygen is formed (for example by exposure of the photosensitizer photofrin to light) which then reacts with a histidine residue in the catalase and thus, inactivates it. This is schematically illustrated in FIG. 3.
However, formation of singlet oxygen can also be achieved by modulation of the NO metabolism of the cells. This is schematically illustrated in FIG. 4. Inhibition of cellular arginase (1), addition of arginine, inhibition of the NO dioxygenase (NOD) (2) or the associated cytochrome P450-dependent oxidoreductase (POR) (3) lead to a sharp increase in the available NO concentration. Finally, by complex amplification steps this results in the formation of extracellular singlet oxygen that inactivates catalase. In EPA 07.870201.6 there is disclosed a method for identifying compounds that induce tumor apoptosis by inactivation of catalase on the surface of tumor cells.
Further, it was described that by manipulation of the NO metabolism and the simultaneous enhancement of the NOX activity a remarkable synergistic effect can be achieved that leads to a reduction of the required concentrations of the single active substance. This mechanism of action is schematically illustrated in FIG. 5. Making use of the synergistic effect between modulators of the NO and der NADPH oxidase is disclosed in EP 10173500.9 A.
While the above-explained synergy mechanisms always act on different partial paths of the ROS signal paths the present invention relates to the synergistic interaction between one or two partial paths of the ROS signaling and a sub-toxic inhibition of the catalase. These combinations then in total lead to a massive inactivation of the catalase and the subsequent selective cell death of the tumor cells.